Unraveling Adaptation of Pontibacter Korlensis to Radiation and Infertility

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OPEN Unraveling adaptation of Pontibacter korlensis to radiation and infertility in desert received: 13 December 2014 accepted: 27 April 2015 through complete genome and Published: 09 June 2015 comparative transcriptomic analysis

Jun Dai1,*, Wenkui Dai2,*, Chuangzhao Qiu2,*, Zhenyu Yang2, Yi Zhang1, Mengzhou Zhou1, Lei Zhang3, Chengxiang Fang4, Qiang Gao2, Qiao Yang5, Xin Li1, Zhi Wang1, Zhiyong Wang6, Zhenhua Jia1 & Xiong Chen1

The desert is a harsh habitat for flora and microbial life due to its aridness and strong radiation. In this study, we constructed the first complete and deeply annotated genome of the genus Pontibacter (Pontibacter korlensis X14-1T = CCTCC AB 206081T, X14-1). Reconstruction of the sugar metabolism process indicated that strain X14-1 can utilize diverse sugars, including cellulose, starch and sucrose; this result is consistent with previous experiments. Strain X14-1 is also able to resist desiccation and radiation in the desert through well-armed systems related to DNA repair, radical oxygen species (ROS) detoxification and the OstAB and TreYZ pathways for trehalose synthesis. A comparative transcriptomic analysis under gamma radiation revealed that strain X14-1 presents high-efficacy operating responses to radiation, including the robust expression of catalase and the manganese transport protein. Evaluation of 73 novel genes that are differentially expressed showed that some of these genes may contribute to the strain’s adaptation to radiation and desiccation through ferric transport and preservation.

Approximately 10% of the Earth’s terrestrial surface is covered by desert with arid environments, which are characterized as environments with nutrient limitation, desiccation, cycles of extreme temperatures and intense radiation1. Nevertheless, diverse bacterial species have been identified and isolated from this extreme biotope2–5 and have been found to be tolerant to solar radiation through various mechanisms, such as DNA repair, ROS detoxification and protein protection6. Since the radiation-resistant strain Deinococcus radiodurans R1 was isolated 50 years ago, studies of bacterial resistance and tolerance to solar radiation have been mainly performed on the genus Deinococcus7. D. radiodurans is 200-fold and 20-fold more resistant to ionizing radiation and UV

1Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, College of Bioengineering, Hubei University of Technology, Wuhan 430068, China. 2BGI Shenzhen, Shenzhen 518083, China. 3College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China. 4China Center for Type Culture Collection (CCTCC), College of Life Sciences, Wuhan University, Wuhan 430072, China. 5East China Sea fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China. 6BGI Yunnan, Kunming 650228, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to X.C. (email: [email protected])

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P. korlensis Pontibacter P. ros e u s X14-1 P. actiniarum DSM19842 sp. BAB1700 DSM17521 Habitat Desert Aquatic Multiple NA Genome size(Mb) 5.46 4.95 4.54 4.58 GC content(%) 47.3 53.1 50.0 52.6 Total genes 5,037 4,689 4,849 4,260 Coding regions(%) 86.58 86.42 84.97 87.01 Unannotated genes 893 901 507 328 Insertion sequence 4 6 1 0 Prophage 1 0 0 0 Transposase/Integrase 114 28 10 5

Table 1. Comparison of strain X14-1 with other species in genus Pontibacter.

irradiation, respectively, than Escherichia coli8, and the complete genome of D. radiodurans was first published in 19999. To elucidate the extreme resistance phenotype of D. radiodurans R1, various research strategies have been combined10, and three hypotheses regarding DNA repair have been proposed11. The lack of novelty in DNA repair-related genes/proteins and the greater efficiency of specific bacteria to use conventional repair pathways are partially supported by the findings from previous studies12–17. An in-depth analysis of the D. radiodurans R1 genome and its gene expression profile revealed that many undefined genes, including ddrA, ddrB, ddrC, ddrD and pprA, are involved in DNA repair18–21, suggesting that repair functions are encoded by these hypothetical genes. The last hypothesis is that ring-like nucleoids (RNs) contribute to DNA repair22. There are also three assumptions regarding the maintenance of a low ROS concentration in bacte- ria10, most of which are detoxifying and scavenging ROS, including small catalase, superoxide dismutase, and antioxidant molecules, and exhibit an increased Mn(II)/Fe ratio intermediated by manganese complexes11,13,23. Daly and Krisko found that molecules smaller than 3 kDa in the extract of Deinococcus radiodurans R1 can impose antioxidant protection on E. coli proteins23,24. The promotion of metabolic activities with decreased ROS production (e.g., glyoxylate bypass of the TCA cycle21,25) is an alternative to the response to oxidative damage and single antioxidant pathways through high ROS production, which could be inactivated due to redundant ROS-tolerance mechanisms. To lower the ROS, it is also helpful to reduce proteins with Fe-S clusters and the number of respiratory chain enzymes25. In addition to maintaining a low ROS concentration, many other metabolic activities, such as proteolysis and glucose metabolism, contribute to the robustness of D. radiodurans R115,23,26,27. In addition to D. radiodurans R1, additional genome sequences of the genus Deinococcus have been published26,28–32, and comparative analyses have been performed to elucidate the diverse molecular mechanisms and physiological determinants underlying the extreme resistance phenotype33,34. We isolated the strain Pontibacter korlensis X14-1T (X14-1) from the surface layer of a desert in Xinjiang, China, and identified it as a new species of the genus Pontibacter2. This study provides the first complete genome of the genus Pontibacter and attempted to delineate genomic components related to radiation and desiccation resistance in comparison with other species from the genus Pontibacter. A comparative analysis of the gene expression profile under radiation was also conducted to unravel the complicated mechanisms of strain X14-1 involved in its adaptation to the arid environment of the desert. This work will provide referable information for the comprehensive understanding of the evolution and adaptation of the genus Pontibacter as well as various radiation and desiccation resistances. Results Genomic characteristics and phylogeny of strain X14-1. The complete genome sequence of strain X14-1 was produced based on high-quality reads and corrected by read mapping and PCR ver- ification. Strain X14-1 has a larger genome size (5.46 MB) and a lower GC content (47.3%) than three other Pontibacter strains distributed in different species (summarized in Table 1). We found that most of the transposase-related genes are located near genomic islands and next to DNA repair- and ROS detoxification-related genes (Fig. 1), which implied that mobile genetic elements (MGEs) play an important role in the adaptation to radiation and desiccation in the desert. Differences in genome size and MGEs between strain X14-1 and other Pontibacter strains may be attributed to the genomic evolution or gapped assembly of P. actiniarum DSM 19842, Pontibacter sp. BAB1700 and P. roseus DSM 17521. To confirm the phylotype of strain X14-1, we downloaded 40 genomes of the family Cytophagaceae (higher taxonomic classification of the genus Pontibacter) available in the NCBI database. Phylogeny analysis indicated the same results as those previously reported based on the 16S rDNA sequence2, and P. actiniarum DSM 19842 was found to be the most homologous to strain X14-1 (Fig. 2), a finding that is also supported by the following functional analysis.

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Figure 1. Genomic components of P. korlensis X14-1. Distribution of genes, genomic islands, transposases, DNA repair and oxidative response related determinants in the genome of strain X14-1. It indicates that strain X14-1 harbors abundant MGEs, suggestive of high genome plasticity.

Sugar metabolism in strain X14-1. In comparison with three other genomes from the genus Pontibacter, we found that only strain X14-1 harbors genes encoding D-fructokinase, which is essential for sucrose and fructose utilization, and this is consistent with previous experimental results2. Beta-galactosidase, which is essential for strain X14-1 to use lactose as an alternative carbon source by catalyzing lactose to galactose and glucose, is specific to strain X14-1 compared with other Pontibacter strains. Although comparative analysis revealed a common dispersion of cellobiose glucohydrolase in Pontibacter, enzymes responsible for degrading cellulose to cellobiose are only distributed in strain X14-1. Starch could be degraded to amylose and alpha-D-galactose-1-phosphate, which is an intermediate in the production of UDP-glucose that could link pentose and glucuronate interconversion. This is important for the utilization of D-galactose as a carbon resource. Mannose can enter glycolysis through beta-D-fructose-6-phosphate with the help of hexokinase and mannose-6-phosphate isomerase, which could be encoded by genes in strain X14-1. The ability to utilize versatile sugars as described above (Fig. 3) could partly explain how strain X14-1 survives in an infertile desert.

Determinants in the genome for the adaptation of strain X14-1 to the radiation and aridness of the desert. Genes related to DNA repair and the stress response, including ROS detoxification and the osmotic response, were analyzed to understand whether and how strain X14-1 protect itself against to desiccation and radiation. In strain X14-1, recombination repair-related genes are the most abundant, followed by base excision repair (BER) and nucleotide excision repair (NER) (Fig. 4, detailed in Supplementary File 1). There are no specific corresponding genetic determinants for the radiation

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Figure 2. Phylogenetic tree of family Cytophagaceae based on 40 available reference genomes and strain X14-1(highlighted in red). The phylogenetic tree was produced on single copy gene within gene family and drawn by TreeBeST version1.9.2 under bootstrap 1000, implicating taxonomic position of strain X14-1.

resistance of strain X14-1, implying that the robustness of the resistance of strain X14-1 to radiation and desiccation is not attributed to new genes but to the high-efficacy operation of systems relevant to recovery from radiation. Trehalose is a natural product that can form a protective film outside the cell under low and high temperatures, osmotic pressure and aridness, preventing proteins from becoming inactivated. The analyses conducted in this study elucidated that intermediate products from the metabolism of several sugars, such as starch and lactose, in strain X14-1 could be transferred to trehalose through the OstAB and TreYZ pathways, as summarized in a previous publication35 (Fig. 5). Trehalase-encoding genes, which are homologous to the trehalase-like protein-encoding genes of Gramella forsetii KT0803, were also identified, suggesting the trehalose consumption of strain X 14-1.

Responses of strain X14-1 to gamma radiation revealed by RNA-Seq. After radiation, the aver- age survival rate of strain X14-1 ranged from 43.4% to 55.1%, as determined from triplicate analyses. To better understand the specific responses of strain X14-1 to gamma radiation, we determined the expression level of all possible determinants responsible for radiation adaptation and ROS detoxification. BER, NER and homologous recombination-related genes, which are mainly required for recovery from gamma radiation, are apparently upregulated (Supplementary File 2). Catalase-encoding genes exhibit intensive upregulation after radiation, but a similar finding was not obtained for superoxide dismutase, which is also important for ROS detoxification (Supplementary File 2). The expression of the manganese transport protein is 10-fold higher after radiation, and a similar trend was identified for subunit A-F of the multicomponent Na+:H+ antiporter as well as other

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Figure 3. Pathways of sugar utilization for strain X14-1. PGI: Pentose and Glucuronate Interconversion; PPP: Pentose Phosphate Pathway. Arrows with different colors represent utilizing distinct carbon sources: Blue-lactose and starch, orange-glycoside, gray-sucrose, brown-cellulose, black-glucose, red-galactose, green- mannose, purple-fructose.

Figure 4. Genes related to DNA repair and stress response in genus Pontibacter. BER: base excision repair; NER: nucleotide excision repair; DR: direct reversal; RER: recombination repair; ODR: other DNA repair; ORT: other radiation tolerance.

ion-coupled transporters (Supplementary File 2). NADH dehydrogenase I, with the exception of except subunit C, exhibited robust expression in the radiated samples compared with the controls. Ferric uptake regulator and LysR family transcriptional regulator, both of which are transcription factors and have been identified as determinants for oxidative detoxification in the genome, also exhibited increased expression after radiation (Supplementary File 2).

Initial evaluation of unannotated genes with differential expression. Apparent differential expression was found for 394 of 893 unclassified genes as a result of gamma radiation. To explore whether these unknown genes contribute to the adaptation of strain X14-1 to radiation, an alignment of these genes to the conserved domain database (CDD) was conducted. Of the 394 new genes, 73 could be

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Figure 5. Trehalose synthesis of strain X14-1. UDP-Glucose from lactose and galactose (Fig. 3) could be catalyzed to trehalose through trehalose 6-phosphate synthase and trehalose 6-phosphate phosphatase. Glycogen operon protein could degrade starch synthesized from alpha-D-Glucose-6P available from many metabolic pathways to trehalose.

assigned to specific domains in CDD, and the corresponding hypothesized functions are summarized in Supplementary File 3, indicating that these 73 genes are commonly found in the FecR, DUF and FlgD_ig (associated with ferric transport and preservation) superfamily. Discussion The desert is characterized by a low diversity of inhabitants due to fluctuations in temperature, aridness, strong radiation and poor nutrition1,36. Elucidating the microbial life in the desert could be important because it could help us understand the dry limit of life and improve our research on extremophiles37. Contributors to radiation resistance and adaptation to poor nutrition in microbes could be applied to improve plant culturing in the desert. This will decrease sand storms mainly induced by the desert and increase arable lands to aid the avoidance of hunger worldwide. Determinants to radiation resistance in microbes residing in the desert could also be industrially applied in cosmetic and soil remediation under radiation. Strain X14-1, which was isolated from the sand surface in the desert, belongs to the poorly under- stood genus Pontibacter, and there are no publications on an in-depth analysis of the Pontibacter genome. In this study, we performed a high-coverage sequencing of strain X14-1 and produced the first complete map, which may be used as a valuable reference to promote research on the genus Pontibacter. A detailed annotation of the strain X14-1 genome in combination with a comparative transcriptomic analysis was conducted to understand how strain X14-1 utilizes diverse alternative nutrition sources and recovers from desiccation and strong radiation. Previous studies have revealed that strain X14-1 can utilize versatile carbon sources2, and this finding is supported by the reconstruction of the metabolic pathways of different sugars. D-fructokinase, beta-galactosidase, endoglucanase and beta-glucosidase are key for the utilization of sucrose, fructose, lactose, cellulose and glycoside, respectively. Enzymes that are important for metabolizing starch, galactose and mannose were also found in strain X14-1. In addition to their robust competence for utilizing diverse available sugars, another interesting finding is that only strain X14-1 and P. actiniarum DSM19842 possess phosphoenolpyruvate (PEP) carboxylase, which is key for CO2 fixation, mainly in plants, through the C4 cycle and may be used to resemble the autotrophic lifestyle in environments with extremely poor nutrition. The fact that most of the hypothetical proteins in strain X14-1 are homologous to those in P. actiniarum DSM19842 also demonstrates the homology of these two Pontibacter strains revealed by the phylogenetic construction (Fig. 2). In addition to the various metabolic pathways required for the utilization of limited resources, strain X14-1 also harbors an intensive arsenal of DNA repair- and stress response-related determinants that allow it to survive in the desert. An in-depth analysis of the genomic components of strain X14-1 revealed possible broad-spectrum contributors to resistance to radiation (Supplementary File 1). This is consistent with the genetic factors found in the genus Deinococcus, and we also found new genes that

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encode resistance agents, indicating supplementary mechanisms to those found in Deinococcus. Based on changes in the annotated genes after gamma radiation, we found that catalases but not dismutase exhibited a marked increase, indicating the robustness of catalase in oxidative detoxification in strain X14-1. An increased Mn(II)/Fe ratio has been demonstrated to play an important role in detoxification from oxidative damage11, and this is also supported by our findings: upregulation of the manganese transport protein, ferric uptake regulator and LysR family transcriptional regulator. The expression of gene gshB encoding glutathione synthase was high during radiation, but glutathione peroxidase and reductase, which are responsible for the circulation of GSS (reduced glutathione) and GSSG (oxidized glutathione) exhibited no change, implying that glutathione is not the intermediate in strain X14-1during radiation resistance. An interesting finding is the robustly elevated expression of transposase during gamma radiation (Supplementary File 2), which implies the potently active role of widely distributed transposases in strain X14-1 for desert adaptation. Some of the conserved domains of upregulated unknown genes could be assigned to the FecR and ferritin-like superfamily. This finding may imply that some unknown genes could contribute to oxidative detoxification because these proteins are related with ferric transport or preservation, but further analysis or experiments are required to confirm this hypothesis. Materials and Methods Strains and culture conditions. All of the strains were obtained from the China Center for Type Culture Collection (CCTCC). P. korlensis X14-1 was grown at 30 °C on marine broth 2216 (Difco). To determine the tolerance of the culture to gamma radiation, the strains were grown in the appropriate liquid medium to the exponential phase, and 2.5 ml of the cell culture was then subjected to 60Co radiation with a continuous dose rate of 1000 Gray/h at room temperature. The control culture cells were not treated with radiation. Samples were collected 3 h after culture with (radiated samples) or without (controls) radiation treatment. Biological triplicates were harvested from each treatment. These samples were immediately frozen in RNAlater (Qiagen, German) and stored at – 80 °C for RNA analysis.

DNA extraction, sequencing and assembly. Total DNA was extracted through the traditional CTAB method, and its quality was tested by Qubit. Sequencing was performed for a 470-bp insert size library (1.65-Gb clean reads with a length of 90 bp) and mate-paired libraries with insert sizes of 2222 bp and 6200 bp (477-Mb clean reads, each with a length of 49 bp), respectively. Clean reads were used to assemble the complete genome sequence as previously described38.

Gene prediction, annotation and comparative analysis. Genes were predicted based on the assembled and confirmed sequence using GLIMMER39–41, which was developed for microorganisms including bacteria, archaea, and viruses. This was also used for the prediction of genes from other three Pontibacter strains downloaded from the NCBI ftp site. The annotations were conducted by assigning the predicted genes to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www. genome.jp/kegg/)42, Cluster of Orthologous Groups of proteins (COG)43,44, and the protein database in NCBI (http://www.ncbi.nlm.nih.gov/protein/) through a BLASTP search (e-value ≤ 1e-5, query coverage ≥ 40%). The strategy described by Tian et al.45 was used to identify the orthologous proteins in four Pontibacter genomes. In the phylogenetic analysis, homologous genes from 41 strains were used to construct the gene family and phylogenetic tree: (i) the gene set was first generated using BLAST (V2.2.23) with the genes from 41 strains; (ii) the gene family was constructed using OrthoMCL (V1.4) with the gene set data; (iii) to construct the phylogenetic tree, single-copy genes in the gene family were screened, and the genes were aligned according to the strains using Muscle (V3.8.31); and (IV) the phylogenetic tree was drawn using TreeBeST (V1.9.2) with 1000 bootstrap replicates.

Identification of MGEs. The prophages in the genome sequences were identified using the Phage Search Tool (PHAST)46. Genomic islands were predicted with multiple methods (IslandPath-DIOMB, SIGI-HMM, and IslandPicker using Islandviewer47–49. We searched repeat sequences with the RepeatMasker program50, and transposases were selected from the annotation results.

RNA-Seq analysis. Three replicates of each group were used for total RNA extraction and mRNA enrichment using the Ribo-ZeroTM kit. The qualified mRNA was sequenced on the HiSeq2000 plat- form, and the expression level of each gene was calculated using the reads per kilo bases per million reads (RPKM)51. The genes that were differentially expressed between the two groups were identified as described by Audicet et al.52. The analysis results with a p-value < 0.05 were corrected by the FDR (false discovery rate), which was set to ≤ 0.001. References 1. Warren Rhodes K. et al. Cyanobacterial ecology across environmental gradients and spatial scales in China’s hot and cold deserts. FEMS microbiology ecology 61, 470–482 (2007). 2. Zhang, L. et al. Pontibacter korlensis sp. nov., isolated from the desert of Xinjiang, China. Int J Syst Evol Microbiol. 58, 1210–1214 (2008). 3. Zhou, Y. et al. Pontibacter akesuensis sp. nov., isolated from a desert soil in China. Int J Syst Evol Microbiol. 57, 321–325 (2007).

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4. Chanal A. et al. The desert of Tataouine: an extreme environment that hosts a wide diversity of microorganisms and radiotolerant bacteria. Environ Microbiol. 8, 514–525 (2006). 5. Rainey F. A. et al. Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample. Appl Environ Microbiol. 71, 5225–5235 (2005). 6. Sabine Matallana-Surget, Ruddy Wattiez. Impact of Solar Radiation on Gene Expression in Bacteria. Proteomes. 1, 70–86 (2013). 7. Anderson A. W., Nordan H. C., Cain R. F., Parrish G. & Duggan D. Studies on a radio-resistant micrococcus. I. Isolation, morphology, cultural characteristics, and resistance to gamma radiation. Food Technol. 10, 575–578 (1956). 8. White, O. et al. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286, 1571–1577 (1999). 9. Battista J. R. Against all odds: the survival strategies of Deinococcus radiodurans. Annu Rev Microbiol. 51, 203–224 (1997). 10. Krisko, A. & M. Radman. Biology of extreme radiation resistance: the way of Deinococcus radiodurans. Cold Spring Harb Perspect Biol. 5, a012765 (2013). 11. Daly M. J. et al. Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306, 1025– 1028 (2004). 12. Daly, M. J. et al. In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans. J Bacteriol. 176, 3508–3517 (1994). 13. Makarova K. S., Aravind L., Daly M. J. & Koonin E. V. Specific expansion of protein families in the radioresistant bacterium Deinococcus radiodurans. Genetics 108, 25–34 (2000). 14. Makarova K. S. et al. Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev. 65, 44–79 (2001). 15. Omelchenko, M. V. et al. Comparative genomics of Thermus thermophilus and Deinococcus radiodurans: divergent routes of adaptation to thermophily and radiation resistance. BMC Evol Biol. 5, 57 (2005). 16. Slade D., Lindner A. B., Paul G. & Radman M. Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans. Cell 136, 1044–1055 (2009). 17. Daly, M. J. & K. W. Minton. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans. J. Bacteriol. 178, 4461–4471 (1996). 18. Tanaka M. et al. Analysis of Deinococcus radiodurans’s transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance. Genetics. 168, 21–33 (2004). 19. Harris D. R. et al. Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol. 2, e304 (2004). 20. Narumi I. et al. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol Microbiol. 54, 278–285 (2004). 21. Liu Y. et al. Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proc Natl Acad Sci. 100, 4191–4196 (2003). 22. Levin-Zaidman, S. et al. Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance? Science 299, 254– 256 (2003). 23. Daly M. J. et al. Small-molecule antioxidant proteome- shields in Deinococcus radiodurans. PLoS ONE 5, e12570 (2010). 24. Krisko A. Radman M. Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans. Proc Natl Acad Sci. 107, 14373–14377 (2010). 25. Ghosal D. et al. How radiation kills cells: Survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress. FEMS Microbiol Rev. 29, 361–375 (2005). 26. de Groot, A. et al. Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti. PLoS Genet. 5, e1000434 (2009). 27. Zhang C., Wei J., Zheng Z., Ying N., Sheng D. & Hua Y. Proteomic analysis of Deinococcus radiodurans recovering from g-irradiation. Proteomics. 5, 138–143 (2005). 28. Pukall, R. et al. Complete genome sequence of Deinococcus maricopensis type strain (LB-34). Stand Genomic Sci. 4, 163–172 (2011). 29. Copeland, A. et al. Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T)). Stand Genomic Sci. 6, 240–250 (2012). 30. Makarova, K. S. et al. Deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks. PLoS One 2, e955 (2007). 31. Mahato, N. K. et al. Draft Genome Sequence of Deinococcus sp. Strain RL Isolated from Sediments of a Hot Water Spring. Genome Announc. 2, e00703-14 (2014). 32. Hu, Y. et al. Draft Genome Sequence of Deinococcus xibeiensis R13, a New Carotenoid-Producing Strain. Genome Announc. 1, e00987-13 (2013). 33. Blasius M., Hubscher U. & Sommer S. Deinococcus radiodurans: what belongs to the survival kit? Crit Rev Biochem Mol Biol. 43, 221–238 (2008). 34. Cox M. M. & Battista J. R. Deinococcus radiodurans—the consummate survivor. Nat Rev Microbiol. 3, 882–892 (2005). 35. Ruhal, R. et al. Trends in bacterial trehalose metabolism and significant nodes of metabolic pathway in the direction of trehalose accumulation. Microb Biotechnol. 6, 493–502 (2013). 36. Wierzchos, J. et al. Microorganisms in desert rocks: the edge of life on Earth. Int Microbiol. 15, 173–183 (2012). 37. Navarro-González et al. Mars-like soils in the Atacama Desert, Chile, and the dry limit of microbial life. Science 302, 1018–1021 (2003). 38. Rohde, H. et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med. 365, 718–724 (2011). 39. Delcher, A. L. et al. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27, 4636–4641 (1999). 40. Salzberg, S. L. et al. Microbial gene identification using interpolated Markov models.Nucleic Acids Res. 26, 544–548 (1998). 41. Delcher, A. L. et al. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23, 673–679 (2007). 42. Kanehisa, M. et al. From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 34, 354–357 (2006). 43. Tatusov, R. L. et al. A genomic perspective on protein families. Science 278, 631–637 (1997). 44. Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41 (2003). 45. Tian, C. F. et al. Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations. Proc Natl Acad Sci. 109, 8629–8634 (2012). 46. Zhou, Y. et al. PHAST: a fast phage search tool. Nucleic Acids Res. 39, W347–W352 (2011). 47. Hsiao, W. et al. IslandPath: aiding detection of genomic islands in prokaryotes. Bioinformatics. 19, 418–420 (2003). 48. Waack, S. et al. Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models. BMC Bioinformatics 7, 142 (2006). 49. Langille, M. G. et al. Evaluation of genomic island predictors using a comparative genomics approach. BMC Bioinformatics 9, 329 (2008). 50. Saha, S. et al. Empirical comparison of ab initio repeat finding programs.Nucleic Acids Res. 36, 2284–2294 (2008). 51. Mortazavi, A. et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 5, 621–628 (2008). 52. Audic, S. & J. M. Claverie. The significance of digital gene expression profiles. Genome Res. 7, 986–995 (1997).

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Acknowledgements We would like to thank Xiaohui Li, Hongjuan Wang and Yongfeng Liu for coordination during this project. This research was supported by the National Natural Science Foundation of China (No: 31300003), the Hubei Provincial Natural Science Foundation (2015CFB679) and Research Fund for the Doctoral Program of Hubei University of Technology (No: BSQD13003). Author Contributions X.C. managed the project. C.F. and Z.W. offered the strain and designed the experiments. Y.Z., L.Z., Q.Y., X.L., Z.W., M.Z. and Z.J. performed experiments and prepared the DNA and RNA samples. Z.Y. and Q.G. performed sequencing and assembly. C.Q. and W.D. performed other bioinformatics analysis in this work. W.D., C.Q. and J.D. wrote the paper. All authors reviewed this manuscript.

Accession codes: Assembly result has been deposited in GenBank nucleotide core database under the accession number CP009621. Clean reads for six transcriptome samples could be found through accession number SRP049974. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Dai, J. et al. Unraveling adaptation of Pontibacter korlensis to radiation and infertility in desert through complete genome and comparative transcriptomic analysis. Sci. Rep. 5, 10929; doi: 10.1038/srep10929 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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